公开(公告)号：US5362628A

公开(公告)日：1994-11-08

申请号：US26633

申请日：1993-03-05

申请人： Richard P. Haugland ,  Yu-Zhong Zhang ,  Ram Sabnis ,  Nels A. Olson ,  John J. Naleway ,  Rosaria P. Haugland

发明人： Richard P. Haugland ,  Yu-Zhong Zhang ,  Ram Sabnis ,  Nels A. Olson ,  John J. Naleway ,  Rosaria P. Haugland

IPC分类号： C12Q1/04 ,  C12Q1/34 ,  G01N33/535 ,  C12Q1/00 ,  A61K37/02 ,  C07H15/24 ,  G01N33/40

CPC分类号： C12Q1/04 ,  C12Q1/34 ,  G01N33/535 ,  C12Q2334/00 ,  C12Q2334/20 ,  C12Q2334/40 ,  C12Q2337/20 ,  G01N2333/924 ,  Y10S530/802

摘要： The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the formXR-REPORTER-BLOCKwherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate,-REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properities different from those of the substrate, andXR-- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R--).After the substrate enters the cells, the analyte removes BLOCK to make REPORTER detectable by the change in spectral properties, and the haloalkyl XR reacts with the intracellular thiol to form the thioether conjugate inside the cells, which is well-retained in the cells.

摘要翻译： 本发明提供了通过仅在存在特定酶的完整细胞中改善可检测报道分子的保留来分析细胞中的代谢活性的方法。 特别地，涉及将报道分子的卤代烷基取代的衍生物与含细胞内半胱氨酸的肽结合的两部分方法的改进的保留结果，同时解除报道分子的阻断。 用于分析细胞代谢活性的方法涉及使用具有XR-REPORTER-BLOCK形式的底物，其中-BLOCK是通过特定分析物的作用被选择为可去除的基团，以产生与底物的不同的REPORTER光谱性质 -REPORTER-是一个分子，当不再通过BLOCK-REPORTER键与BLOCK结合时，具有与底物不同的光谱性质，XR-是可以与胞内硫醇（ZSH）共价反应的卤代烷基部分， 以形成硫醚缀合物（ZSR-）。 在底物进入细胞后，分析物除去BLOCK以通过光谱性质的变化使REPORTER可检测到，并且卤代烷基XR与细胞内硫醇反应以在细胞内形成硫醚缀合物，其在细胞中良好保留。